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<h4>PURPOSE</h4><p>To assess ultrastructural stromal modifications in porcine corneas after riboflavin and ultraviolet A (UVA) exposure using immunofluorescence confocal imaging.</p> <h4>METHODS</h4><p>Twenty-five freshly enucleated porcine eyes were enrolled in the study. Five eyes served as control (group I). Twenty eyes had their epithelium removed (groups I, II, IV, and V) and five eyes had their epithelium intact (group III). Groups II and III were cross-linked with riboflavin 0.1% solution (10 mg riboflavin-5-phosphate in 10 mL 20% dextran-T-500) and exposed to UVA (365 nm, 3 mW/cm²) for 30 minutes. Group IV included five eyes soaked with riboflavin without posterior irradiation, and group V included five eyes irradiated, without previous exposure to riboflavin. Ultra-thin sections (8 µm) of the corneas were stained with anti-collagen I and DAPI and their fluorescence was revealed under confocal microscopy.</p> <h4>RESULTS</h4><p>Only the cross-linked corneas (group II) showed a pronounced, highly organized anterior fluorescence zone of 182.5±22.5 µm. Using DAPI staining, an anterior and concentrated displacement of cell nuclei due to collagen compaction was observed after cross-linking (group II). No structural changes were observed in all other groups.</p> <h4>CONCLUSIONS</h4><p>The cross-linking treatment effect can be directly visualized using confocal fluorescence imaging, allowing for a quantitative analysis. Cross-linked corneas showed a pronounced and limited anterior zone of organized collagen fibers, which was not observed in the other groups. Treatment of the cornea with riboflavin and UVA without previous deepithelialization did not induce any cross-linking effect. Consequently, to facilitate diffusion of riboflavin throughout the corneal stroma, the epithelium should be removed as an important initial step in the treatment. [<cite>J Refract Surg.</cite> 2008;24:S715-S719.]</p> <h4>ABOUT THE AUTHORS</h4> <p>From the Departments of Ophthalmology (Bottós, Regatieri, Lima-Filho, Schor, Chamon) and Biochemistry (Dreyfuss, Regatieri, Nader), Federal University of São Paulo, Brazil.</p> <p>The authors have no financial interest in the materials presented herein.</p> <p>Presented in part at the International Congress of Corneal Cross-Linking; December 7-8, 2007; Zurich, Switzerland.</p> <p>Correspondence: Wallace Chamon, MD, Av Indianopolis, 1797, São Paulo, SP 04063-003, Brazil. E-mail: <A href="mailto:visus@pobox.com"> visus@pobox.com</a></p>

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Corneal collagen cross-linking (CXL) has been described as a promising therapy for keratoconus. According to standard CXL protocol, epithelium should be debrided before treatment to allow penetration of riboflavin into the corneal stroma. However, removal of the epithelium can increase procedure risks. In this study we aim to evaluate stromal penetration of a biocompatible riboflavin-based nanoemulsion system (riboflavin-5-phosphate and riboflavin-base) in rabbit corneas with intact epithelium. Two riboflavin nanoemulsions were developed. Transmittance and absorption coefficient were measured on corneas with intact epithelia after 30, 60, 120, 180, and 240 minutes following exposure to either the nanoemulsions or standard 0.1% or 1% riboflavin-dextran solutions. For the nanoemulsions, the epithelium was removed after measurements to assure that the riboflavin had passed through the hydrophobic epithelium and retained within the stroma. Results were compared to de-epithelialized corneas exposed to 0.1% riboflavin solution and to the same riboflavin nanoemulsions for 30 minutes (standard protocol). Mean transmittance and absorption measured in epithelialized corneas receiving the standard 0.1% riboflavin solution did not reach the levels found on the debrided corneas using the standard technique. Neither increasing the time of exposure nor the concentration of the riboflavin solution from 0.1% to 1% improved riboflavin penetration through the epithelium. When using riboflavin-5-phosphate nanoemulsion for 240 minutes, we found no difference between the mean absorption coefficients to the standard cross-linking protocol (p = 0.54). Riboflavin nanoemulsion was able to penetrate the corneal epithelium, achieving, after 240 minutes, greater stromal concentration when compared to debrided corneas with the standard protocol (p = 0.002). The riboflavin-5-phosphate nanoemulsion diffused better into the stroma than the riboflavin-base nanoemulsion.

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YFP(+) BMCs in thy1-YFP mice have immunophenotypic features of MDSCs. They secrete NGF and promote neuroregeneration. Their immunosuppressive and neurotrophic actions are preserved after corneal infiltration. These findings increase our understanding of the beneficial roles played by leukocyte trafficking in the cornea and may lead to therapeutic strategies that use NGF-secreting myeloid cells to repair diseased or injured neurons.